Administration of angiocidin for the treatment of cancer

ABSTRACT

Methods are presented for the therapeutic administration of angiocidin in the treatment of cancers such as glioma, breast cancer, and leukemia. Methods are also presented for inducing growth arrest and/or apoptosis of tumor cells, as well as inducing differentiation of tumor cells to inhibit tumorigenicity and to confer a non-tumor or healthy phenotype.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. Ser. No.13/130,898, filed on Jul. 28, 2011, which is a U.S. national stage ofthe PCT International Application No. PCT/US2009/065301, filed Nov. 20,2009, which claims priority to U.S. Provisional Application Nos.61/145,836, filed Jan. 20, 2009, and 61/117,745, filed Nov. 25, 2008.Each of these applications is incorporated by reference herein, in itsentirety and for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R01 CA88931awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety.

Solid human tumors are often infiltrated by host immune cells, which canhave a diverse effect on tumor progression. Among other cell types,macrophages are known to be a major component in the leukocyteinfiltrate in tumors. These tumor-associated macrophages, (TAMs), have acomplex dual function in their interactions with neoplastic cells(Mantovani, et al., Trends Immunol. 23: 549-555, 2002). First, TAMsstimulate cell destruction through antigen presentation to T-cells,which induces cytotoxic T-cells to kill tumor cells bearing thepresented antigen. In contrast, TAMs also promote cell proliferation andangiogenesis, thus affecting tissue growth.

These contradictory effects can be explained in terms of the “macrophagebalance hypothesis,” which asserts that the outcome of the interactionbetween macrophages and neoplastic cells depends on the number ofmacrophages recruited to the tumor microenvironment and their state ofactivation (Mantovani, et al., Immunol. Today 13: 265-270, 1992; Bingle,et al., J. Pathol. 196: 254-265, 2002; Nesbit, et al., J. Immunol. 166:6483-6490). Nesbit, et al. have shown that, in a mouse model, highlevels of monocyte chemoattractant protein-1 (MCP-1) secreted bymelanoma cells is associated with massive monocyte/macrophageinfiltration into the tumor mass, leading to destruction of the tumorwithin a few days. However, low levels of secreted MCP-1 stimulatedangiogenesis and tumor growth. Furthermore, ex vivo-grown cytotoxicmacrophages that recognize and destroy tumor cells, but not normalcells, are effective in murine models of metastasizing tumors.(Andreesen, et al., J. Leukocyte Biol. 64: 419-426, 1998). Accordingly,immunomodulation, in particular regulating macrophage activity, haspotential as a therapeutic strategy for the treatment of tumors,secondary metastasis, and other disorders.

Angiocidin is a protein, originally isolated from lung carcinoma, thatis overexpressed in many tumor systems (Zhou et al., J. Cell. Biochem.92: 125-146, 2004; Poon, et al., Clin. Cancer Res. 12: 4150-4157, 2004).Angiocidin is a receptor for thrombospondin-1 and is a potent inhibitorof angiogenesis and tumor cell proliferation (US2003/0180295; Zhou etal., J. Cell. Biochem. 92: 125-146, 2004). These functions of angiocidinare mediated by α2β1 integrin (Sabherwal, et al., Exp. Cell Res. 312:443-453, 2006). In addition, angiocidin has important immunomodulatoryeffects on monocytes that can affect the course of disease.

SUMMARY OF THE INVENTION

The invention features methods for treating tumors such as glioma,breast cancer, and leukemia. The methods generally compriseadministering to a subject having a glioma, breast cancer, or leukemiaan amount of angiocidin effective to treat the glioma, breast cancer, orleukemia. The angiocidin can be administered to the subject as acomposition comprising a pharmaceutically acceptable carrier. The amountof angiocidin administered to the subject can vary according to the typeof tumor, or other variables, but generally will be a dose of about0.001 to 10 mg/kg body weight of the subject.

In some aspects, the methods further comprise administering to thesubject an effective amount of a growth factor such as fibroblast growthfactor-2 (FGF-2), nerve growth factor (NGF), brain-derived neural factor(BDNF), neurotropin-3 (NT-3), epidermal growth factor (EGF), or stemcell growth factor (SCF). The angiocidin can complex with the growthfactor. In some aspects, the angiocidin-growth factor complex can inducegrowth arrest of the tumor cells, such as the glioma, breast cancer, orleukemia cells. In some aspects, the angiocidin-growth factor complexcan promote healthy stem cell differentiation at or proximal to the siteof the glioma or breast cancer, or in the blood or the bone marrow.

The invention also features methods for arresting growth of tumor cells.The methods generally comprise contacting the tumor cell with an amountof angiocidin effective to arrest growth of the tumor cell. In someaspects, subsequent to or concomitant with the growth arrest, the tumorcell undergoes apoptosis. The methods are preferably used to arrest thegrowth of glioma cells, breast cancer cells, leukemia cells, andmelanoma cells. In some aspects, the angiocidin can be complexed with agrowth factor such as fibroblast growth factor-2 or nerve growth factor.

The invention also features methods for inducing differentiation ofleukemia cells. The methods generally comprise contacting the leukemiacell with an amount of angiocidin effective to induce differentiation ofthe leukemia cell. Leukemia cell differentiation preferably inhibits thetumorigenicity of the leukemia cell. In some aspects, induced leukemiacell differentiation confers a non-cancerous phenotype.

The invention also features methods for treating tumors that express agrowth factor receptor. The methods generally comprise administering toa subject having a tumor that expresses a receptor for fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor anamount of angiocidin effective to treat the tumor expressing a receptorfor fibroblast growth factor-2, nerve growth factor, brain-derivedneural factor, neurotropin-3, epidermal growth factor, or stem cellgrowth factor. The angiocidin can be administered to the subject as acomposition comprising a pharmaceutically acceptable carrier. The amountof angiocidin administered to the subject can vary according to the typeof tumor, or other variables, but generally will be a dose of about0.001 to 10 mg/kg body weight of the subject. The tumor expressing areceptor for such growth factors can be, for example, glioma, breastcancer, leukemia, or melanoma among others.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a fluorescence micrograph showing the macrophage-likemorphology and formation of focal adhesions by THP-1 cells treated withangiocidin. Cells were reacted with an anti-vinculin antibody and DAPI.

FIG. 2 presents graphs showing the effects of angiocidin-conditionedmedium on THP-1 and PBMC cell migration through FIG. 2A—gelatin, FIG.2B—a HUVEC layer. FIG. 2C shows the effects of a 25 amino acid peptidecomprising SEQ ID NO:2 on adhesion of THP-1 cells.

FIG. 3 shows the effects of angiocidin on the morphology and phenotypeof THP-1 cells. Angiocidin-treated THP-1 cells developed focal adhesionsand exhibited an increase in phosphorylated FAK protein andphosphorylated paxillin (FIGS. 3A and 3B). Angiocidin stimulated mRNAexpression for a number of proteins that are “differentiation statedependent,” including CD36, MARCO, CD14, CD69, and α2-macroglobulin(FIGS. 3C and 3D).

FIG. 4 shows the effects of angiocidin on human PBMC and THP-1 cells.FIG. 4A—Flow cytometric analysis of cellular activation of PBMC.Percentage activation is shown in the upper right quadrant of each plot.FIG. 4B—Integrated density values for inflammatory mediators secreted byTHP-1 cells. FIG. 4C—Percentage increase in each cytokine in culturemedium following treatment with angiocidin. FIG. 4D—Concentration ofMCP-1 in culture medium of THP-1 cells.

FIG. 5 shows the generation of antigen-specific IL-2 producing cells inangiocidin- and/or MBP-treated monocytes challenged with untreated PBMC(FIG. 5A) or PBMC treated with angiocidin (FIG. 5C), MBP (FIG. 5B), orangiocidin+MBP (FIG. 5D)

FIG. 6 presents phase-contrast micrographs of BLSC from human bloodfollowing treatment with FIG. 6A—angiocidin, FIG. 6B—bFGF, or FIG.6C—bFGF+EGF.

FIG. 7 presents graphs showing the effects of angiocidin onproliferation of melanoma stem cells after FIG. 7A—one week, or FIG.7B—two weeks.

FIG. 8 shows the effect of angiocidin on PC12 neurite growth. FIG.8A—angiocidin promotes NGF-mediated differentiation of PC12 cells, FIG.8B—neurite outgrowth quantitation.

FIG. 9 shows the effect of angiocidin on the growth of glioma stemcells.

FIG. 10 shows direct binding of angiocidin to adsorbed nerve growthfactor (NGF) and fibroblast growth factor-2 (FGF-2). FIG. 10A—the boundbiotinylated angiocidin was detected with streptavidin-coupled horseradish peroxidase and developed with the colorimetric substrate ultra3,3′,5,5′-tetramethylbenzidine (TMB). FIG. 10B—to determine thatbiotinylated angiocidin binds specifically, it was shown that bindingwas competed with unbiotinylated angiocidin.

FIG. 11 shows the effect of angiocidin and NGF on MDA MB231 breastcarcinoma proliferation.

FIG. 12 shows that MDA-MB-231 human breast tumor cells transfected withangiocidin to over-express angiocidin formed smaller colonies in softagar and developed smaller tumors when injected subcutaneously intoathymic mice. FIG. 12A—paraformaldehyde-fixed cells grown in culture andstained with anti-angiocidin antibody and developed with DAB (100×),FIG. 12B—Western blot of cells grown in panel A, FIG. 12C—colonies grownin soft agar as seen by phase contrast phase contrast (50× mag), FIG.12D—tumor volumes of cells grown in mice (n=6 per group±std).

FIG. 13 shows that angiocidin differentiates CD34+CD38-Leukemic stemcells. Cells were photographed at 200× magnification using Hoffmaninterference microscopy.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural unless the content clearlydictates otherwise. Thus, for example, reference to “a cell” includes acombination of two or more cells, and the like.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of angiocidin, a growthfactor, or composition, including those as described herein, effectiveto achieve a particular biological result such as, but not limited to,biological results disclosed, described, or exemplified herein. Suchresults may include, but are not limited to, the treatment of tumors ina subject, as determined by any means suitable in the art.

Except when noted, “subject” or “patient” are used interchangeably andrefer to mammals such as humans and non-human primates, as well ascompanion, farm, or experimental animals such as rabbits, dogs, cats,rats, mice, horses, cows, pigs, and the like. Humans are most preferred.

Recent studies have shown that angiocidin stimulates important cellularimmune system responses. Angiocidin activates monocytes, stimulating theproduction of proinflammatory cytokines and chemokines and stimulatingantigen presentation to T-cells, which leads to the formation ofcytotoxic T-cells. Furthermore, angiocidin induces the differentiationof monocytes to macrophages. Because of these activities, angiocidin haspreviously unknown therapeutic value for the treatment of a number ofdisorders that are regulated, at least in part, by the immune system,including tumor cell metastasis, leukemia, wound healing, skin damage,and autoimmune disorders such as multiple sclerosis.

The ability of cancer cells to metastasize to distant sites in the bodyis responsible for the majority of cancer-related deaths. The surgicalremoval of a primary tumor can stimulate metastasis and secondary tumorformation, suggesting that primary tumors secrete factors that inhibitsecondary metastases. Two such factors, endostatin and angiostatin, havebeen described (Dong, et al., Cell 88: 801-810, 1997; Yokoyama, et al.,Cancer Research 60: 2190-2196, 2000). Angiocidin appears to be anotherinhibitory factor of tumor origin that can prevent or treat secondarymetastasis by stimulating macrophage differentiation and recruitingcytotoxic T-cells to the metastatic site for tumor cell destruction. Asa result, secondary tumor development is prevented. Accordingly, in oneembodiment of the invention, a composition comprising angiocidin and apharmaceutically acceptable carrier is administered to a subject in needthereof to suppress or prevent metastasis. Angiocidin can also beadministered directly to the surgical site following removal of aprimary tumor. Particular tumor tissue types that could be treated inthis manner include, but are not limited to, breast, colon, lung,prostate, skin, oral, nasal, esophageal, stomach, liver, pancreas,glioma, head, neck, ovarian and uterine.

As described in detail in Examples 2 and 3, angiocidin activatesmonocytes and, unexpectedly, induces the differentiation of monocytes tomacrophages. Accordingly, in another embodiment of the invention,angiocidin is administered to a subject in need thereof for thetreatment of leukemia, which is characterized by the uncontrolledproliferation of leukemic blast cells. Angiocidin induces leukemic cellsto differentiate and cease proliferation, thereby correcting themalignant phenotype. This “differentiation therapy” provides a method tomanage the disease without debilitating cytotoxic chemotherapy.Similarly, Example 7 demonstrates that angiocidin inhibits theproliferation of stem cells that give rise to melanoma.

In another embodiment, angiocidin is administered to develop andstimulate antigen-presenting macrophages to recruit T-cells to sites ofinfection. Angiocidin stimulates these T-cells to produce substances,e.g., IL-2 and IL-12, that lyse tumor and foreign cells and raise animmune response against the infective organism. Thus, angiocidin-inducedmacrophage differentiation and stimulation can be used to treat andprevent viral, bacterial, and protozoal infections. These may include,but are not limited to, herpes simplex virus I and II infections,varicella-zoster virus/Ellen, bovine papilloma virus, and humanimmunodeficiency virus, hepatitis B and C, influenza, and tuberculosis.

Blastomere-like stem cells (BLSC), totipotent stem cells found in humanperipheral blood, will also differentiate in response to angiocidin(Example 6). BLSC were developed by Moraga Biotechnology Corp., LosAngeles, Calif., and are described further in WO/2007/100845. Astotipotent cells, BLSC have the ability to differentiate into mostmammalian cell types. These native (i.e., not tumor-derived) stem cellsare not cancerous when implanted into animals, but can incorporate intoall tissues undergoing repair and will proliferate until stimulated bymicroevironmental cues from cells of a specific tissue to differentiateinto that particular cell type. BLSC are capable of generating tissuederived from all three germ layer lineages, including germ cells, inresponse to a combination of general and specific differentiationsignals. These and other characteristics of BLSC are discussed inmoragabiotech.com/techblscs.htm. These results indicate that angiocidinhas in vitro and in vivo uses as a general induction agent in tissueregeneration, neurogenesis, wound healing or tissue repair.Additionally, angiocidin has applications in cosmetics forneocollagenesis and skin rejuvenation.

The invention provides methods for treating tumors that express areceptor for fibroblast growth factor-2, nerve growth factor,brain-derived neural factor, neurotropin-3, epidermal growth factor, orstem cell growth factor. The methods generally comprise administering toa subject having a tumor that expresses a receptor for fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor anamount of angiocidin effective to treat the tumor expressing a receptorfor fibroblast growth factor-2, nerve growth factor, brain-derivedneural factor, neurotropin-3, epidermal growth factor, or stem cellgrowth factor. The angiocidin can be administered to the subject as acomposition comprising a pharmaceutically acceptable carrier. The amountof angiocidin administered to the subject can vary according to the typeof tumor, or other variables, but generally will be a dose of about0.001 to 10 mg/kg body weight of the subject. The tumor expressing areceptor for fibroblast growth factor-2, nerve growth factor,brain-derived neural factor, neurotropin-3, epidermal growth factor, orstem cell growth factor can be, for example, glioma, breast cancer,leukemia, or melanoma among others.

The invention provides methods for treating a glioma in a subject. Themethods generally comprise administering to the subject an amount ofangiocidin effective to treat the glioma. The angiocidin can beadministered in a composition comprising angiocidin and apharmaceutically acceptable carrier. The angiocidin can be administeredto the subject in a dose of about 0.001 to 10 mg/kg body weight.

The methods may further comprise administering to the subject aneffective amount of a growth factor such as fibroblast growth factor-2(FGF-2), nerve growth factor (NGF), brain-derived neural factor (BDNF),neurotropin-3 (NT-3), epidermal growth factor (EGF), and stem cellgrowth factor (SCF), or any other growth factor having a basicisoelectric point. FGF-2 and NGF are most preferred. The growth factorscan be administered in the same composition with angiocidin. The growthfactors can be administered in a different composition, which comprisesthe growth factor and a pharmaceutically acceptable carrier. The growthfactors can be administered at the same time, in advance of, or afterthe administration of angiocidin.

In some aspects, the angiocidin complexes with the fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor, andthe complex induces growth arrest of the glioma cells. In some aspects,the complex promotes healthy stem cell differentiation at or proximal tothe site of the glioma.

The invention provides methods for treating breast cancer in a subject.The methods generally comprise administering to the subject an amount ofangiocidin effective to treat the breast cancer. The angiocidin can beadministered in a composition comprising angiocidin and apharmaceutically acceptable carrier. The angiocidin can be administeredto the subject in a dose of about 0.001 to 10 mg/kg body weight.

The methods may further comprise administering to the subject aneffective amount of a growth factor such as fibroblast growth factor-2,nerve growth factor, brain-derived neural factor, neurotropin-3,epidermal growth factor, or stem cell growth factor. The growth factorscan be administered in the same composition with angiocidin. The growthfactors can be administered in a different composition, which comprisesthe growth factor and a pharmaceutically acceptable carrier. The growthfactors can be administered at the same time, in advance of, or afterthe administration of angiocidin.

In some aspects, the angiocidin complexes with the fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor, andthe complex induces growth arrest of the breast cancer cells. In someaspects, the complex promotes healthy stem cell differentiation at orproximal to the site of the breast cancer.

The invention provides methods for treating leukemia in a subject. Themethods generally comprise administering to the subject an amount ofangiocidin effective to treat the leukemia. The angiocidin can beadministered in a composition comprising angiocidin and apharmaceutically acceptable carrier. The angiocidin can be administeredto the subject in a dose of about 0.001 to 10 mg/kg body weight.

The methods may further comprise administering to the subject aneffective amount of a growth factor such as fibroblast growth factor-2,nerve growth factor, brain-derived neural factor, neurotropin-3,epidermal growth factor, or stem cell growth factor. The growth factorscan be administered in the same composition with angiocidin. The growthfactors can be administered in a different composition, which comprisesthe growth factor and a pharmaceutically acceptable carrier. The growthfactors can be administered at the same time, in advance of, or afterthe administration of angiocidin.

In some aspects, the angiocidin complexes with the fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor, andthe complex induces growth arrest of the leukemia cells. In someaspects, the complex promotes healthy stem cell differentiation in theblood or in the bone marrow.

The invention also provides methods for arresting the growth of a tumorcell. The methods generally comprise contacting the tumor cell with anamount of angiocidin effective to arrest growth of the tumor cell. Insome aspects, the tumor cell will undergo apoptosis subsequent to itsexposure to angiocidin, and the apoptosis may begin subsequent to thegrowth arrest. Angiocidin can be used to arrest the growth of any tumorcell, with glioma cells, breast cancer cells, leukemia cells, andmelanoma cells being highly preferred. In some aspects, the angiocidincan be complexed with a growth factor such as fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor, orother growth factors with similar structures to these growth factors. Itis believed that the angiocidin can potentiate the action of such growthfactors. The methods can be carried out in vitro or in vivo.

The invention also provides methods for inducing differentiation of aleukemia cell. The methods generally comprise contacting the leukemiacell with an amount of angiocidin effective to induce differentiation ofthe leukemia cell. The differentiation of the leukemia cell inducesgenotypic and phenotypic changes that inhibit, and that are capable ofreversing the tumorigenicity of the leukemia cell, for example, makingthe leukemia cell less cancerous. For example, differentiation ofleukemic cells may result in downregulation and expression of theoncogene BCL-2. Cells can also be considered less cancerous if they failto grow in soft agar and display a less invasive phenotype, or arecapable of phagocytosis. In some aspects, the differentiation inducesthe leukemia cell to confer a non-cancerous phenotype, for example, aphenotype characterized by a cell capable of anchorage-dependent growthwith a well-defined cytoskeleton. It is believed that angiocidin mayinduce leukemic cells to lose their potential to initiate a tumor whenengrafted into an immunocompromised host. It is believed that thedifferentiation of a leukemia cell by angiocidin can be enhanced bycomplexing the angiocidin with a growth factor such as FGF-2, NGF, BDNF,NT-3, EGF, and SCF. The differentiated phenotype in leukemia can be acell expressing molecules normally expressed by terminallydifferentiated immune cells such as CD14, a macrophage marker, andmolecules that can present antigens to T cells. In addition, suchphenotypes include that differentiated cells no longer can self-renew,have a limited lifespan, downregulate oncogenes, cannot metastasize, andare contact inhibited in vitro.

A method of treating a human or other mammal in need thereof withangiocidin comprising administering to the human or other mammal atherapeutically effective dose of angiocidin is also provided. A methodfor activating or inducing the differentiation of native stem cellscomprising administering angiocidin to one or more native stem cells,wherein the stem cells are activated or induced to differentiate intolineages of the three germ layers. A method for inducing differentiationof tumor-derived stem cells comprising administering angiocidin totumor-derived stem cells in vivo or in vitro, wherein the tumor-derivedcancer stem cells are induced to differentiate into cells which losetheir ability to hyperproliferate and possess a gene expression profileof terminally differentiated cells, similar to normal cells encompassingthe tumor.

Angiocidin may be administered to a subject by any appropriate means,such as enteral, parenteral, transdermal, or by direct injection orapplication to a diseased site. Metastasis may be inhibited by applyingangiocidin directly to the surgical site following removal of a primarytumor. Dosage is preferably determined by a physician based on thedisease to be treated and the physiological status of the subject,however, the dose may range from about 0.001-10 mg/kg body weight, andis preferably at about 0.01 to 1.0 mg/kg body weight. A preferred dosagerange for leukemia is about 0.01-0.1 mg/kg. For in vitro use, angiocidinmay be applied at about 1-50 μg/mL, preferably at about 5-20 fag/ml.

The effective amount of the angiocidin may be dependent on any number ofvariables, including without limitation, the species, breed, size,height, weight, age, overall health of the subject, the type offormulation, the mode or manner or administration, the type and/orseverity of the particular cancer being treated, the stage of cancer,the extent of metastasis, and the like. The appropriate effective amountcan be routinely determined by those of skill in the art using routineoptimization techniques and the skilled and informed judgment of thepractitioner and other factors evident to those skilled in the art.Preferably, a therapeutically effective dose of the angiocidin willprovide therapeutic benefit without causing substantial toxicity to thesubject.

Toxicity and therapeutic efficacy of the angiocidin formulations can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Agents or compositions which exhibit large therapeuticindices are preferred. The data obtained from cell culture assays andanimal studies can be used in formulating a range of dosage for use inthe subject. The dosage of such agents or compositions lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.

For any angiocidin composition used in the methods of the invention, thetherapeutically effective dose can be estimated initially from in vitroassays such as cell culture assays. For example, a dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 as determined in cellculture. Such information can be used to more accurately determineuseful doses in a specified subject such as a human. The treatingphysician can terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions, and can adjust treatment asnecessary if the clinical response is not adequate in order to improvethe response.

In some aspects, the dose of angiocidin administered to the subject canalso be measured in terms of total amount of drug administered per day.Treatment can be initiated with smaller dosages that are less than theoptimum dose of angiocidin, followed by an increase in dosage over thecourse of the treatment until the optimum effect under the circumstancesis reached. If needed, the total daily dosage may be divided andadministered in portions throughout the day.

Angiocidin is preferably administered as a pharmaceutical formulationwith one or more carriers and/or excipients that are compatible withboth the compounds administered and the subject to whom they areadministered. Angiocidin may be administered alone or in combinationwith another active ingredient. Formulations of angiocidin may bepresented in unit-dose or multi-dose forms and may be prepared by anymethods known in the pharmaceutical art.

Pharmaceutically acceptable carriers can be either solid or liquid.Non-limiting examples of solid form preparations include powders,tablets, pills, capsules, lozenges, cachets, suppositories, dispersiblegranules, and the like. A solid carrier can include one or moresubstances which may also act as diluents, flavoring agents, bufferingagents, binders, preservatives, tablet disintegrating agents, or anencapsulating material. Suitable solid carriers include magnesiumcarbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin,starch, gelatin, acacia, tragacanth, methylcellulose, sodiumcarboxymethyl-cellulose, polyethylene glycols, vegetable oils, agar, alow melting wax, cocoa butter, and the like. Non-limiting examples ofliquid form preparations include solutions, suspensions, syrups,slurries, and emulsions. Suitable liquid carriers include any suitableorganic or inorganic solvent, for example, water, alcohol, salinesolution, buffered saline solution, physiological saline solution,dextrose solution, water propylene glycol solutions, and the like,preferably in sterile form.

Aqueous solutions can be prepared by dissolving the angiocidin and/orgrowth factor in water and adding suitable colorants, flavors,stabilizing and thickening agents as desired. Aqueous suspensions canalso be made by dispersing the finely divided active component in waterwith viscous material, such as natural or synthetic gums, resins,methylcellulose, sodium carboxymethylcellulose, and other well-knownsuspending agents.

Solid forms can be prepared according to any means suitable in the art.For example, capsules can be prepared by mixing the active ingredientsor composition with a suitable diluent and filling the proper amount ofthe mixture in capsules. Tablets are prepared by direct compression, bywet granulation, or by dry granulation. Their formulations usuallyincorporate diluents, binders, lubricants and disintegrators as well asthe compound. Non-limiting examples of diluents include various types ofstarch, cellulose, crystalline cellulose, microcrystalline cellulose,lactose, fructose, sucrose, mannitol, kaolin, calcium phosphate orsulfate, inorganic salts such as sodium chloride and powdered sugar.Powdered cellulose derivatives are also useful. Non-limiting examples oftablet binders include starches, gelatin and sugars such as lactose,fructose, glucose and the like. Natural and synthetic gums are alsoconvenient, including acacia, alginates, methylcellulose,polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcelluloseand waxes can also serve as binders.

A lubricant can be used in a tablet formulation to prevent the tabletand punches from sticking in the die. The lubricant can be chosen fromsuch slippery solids as talc, magnesium and calcium stearate, stearicacid and hydrogenated vegetable oils. Tablet disintegrators aresubstances which swell when-wetted to break up the tablet and releasethe compound, and include starches such as corn and potato starches,clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, woodcellulose, powdered natural sponge, cation-exchange resins, alginicacid, guar gum, citrus pulp, carboxymethyl cellulose, and sodium laurylsulfate. Tablets can be coated with sugar as a flavor and sealant, orwith film-forming protecting agents to modify the dissolution propertiesof the tablet. The compounds may also be formulated as chewable tablets,by using large amounts of pleasant-tasting substances such as mannitolin the formulation, as is now well-established in the art.

Also included are liquid formulations and solid form preparations whichare intended to be converted, shortly before use, to liquid formpreparations. Such liquid forms include solutions, suspensions, syrups,slurries, and emulsions. Liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats or oils); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). These preparations maycontain, in addition to the active agent, colorants, flavors,stabilizers, buffers, artificial and natural sweeteners, dispersants,thickeners, solubilizing agents, and the like. The compositions may bein powder form for constitution with a suitable vehicle such as sterilewater, saline solution, or alcohol, before use.

Permeability enhancers can significantly enhance the permeability oflipophilic and nonlipophilic drugs. Additional forms of chemicalenhancers, such as those enhancing lipophilicity, have been developed toimprove transport when physically mixed with certain therapeutic agentsand provide more predictable absorption. Carriers have also been coupledto pharmaceutical agents to enhance intracellular transport.

Typical permeation enhancers may include bile salts such as sodiumcholate, sodium glycocholate, sodium glycodeoxycholate,taurodeoxycholate, sodium deoxycholate, sodium lithocholatechenocholate, chenodeoxycholate, ursocholate, ursodeoxycholate,hydrodeoxycholate, dehydrocholate, glycochenocholate, taurochenocholate,and taurochenodeoxycholate. Other permeation enhancers such as sodiumdodecyl sulfate (“SDS”), dimethyl sulfoxide (“DMSO”), sodium laurylsulfate, salts and other derivatives of saturated and unsaturated fattyacids, surfactants, bile salt analogs, derivatives of bile salts, orsuch synthetic permeation enhancers as described in U.S. Pat. No.4,746,508 may be used. It is generally believed that bile salts are goodenhancers for hydrophilic drugs and long chain fatty acids, their salts,derivatives, and analogs are more suitable for lipophilic drugs. DMSO,SDS, and medium chain fatty acids (about C-8 to about C-14) their salts,derivatives, and analogs may work for both hydrophilic and lipophilicdrugs.

The permeation enhancer concentration within the dissolvable matrixmaterial may be varied depending on the potency of the enhancer and rateof dissolution of the dissolvable matrix. Other criteria for determiningthe enhancer concentration include the potency of the drug and thedesired lag time. The upper limit for enhancer concentration is set bytoxic effect to or irritation limits of the mucosal membrane.

The compositions may also include a disintegrating agent. Tabletdisintegrators are substances which swell when-wetted to break up thetablet and release the compound, and include starches such as corn andpotato starches, clays, celluloses, aligns, gums, methylcellulose, agar,bentonite, wood cellulose, powdered natural sponge, cation-exchangeresins, alginic acid, sodium alginate, guar gum, citrus pulp,carboxymethyl cellulose, polyvinylpyrrolidone, and sodium laurylsulfate. Acrylic type polymers can also advantageously be used asdisintegrators, including methacrylic copolymers of type C.

The compositions can be formulated for use in topical administration.Such formulations include, e.g., liquid or gel preparations suitable forpenetration through the skin such as creams, liniments, lotions,ointments or pastes, and drops suitable for delivery to the eye, ear ornose.

In some embodiments, the present compositions include creams, drops,liniments, lotions, ointments and pastes are liquid or semi-solidcompositions for external application. Such compositions may be preparedby mixing the active ingredient(s) in powdered form, alone or insolution or suspension in an aqueous or non-aqueous fluid with a greasyor non-greasy base. The base may comprise complex hydrocarbons such asglycerol, various forms of paraffin, beeswax; a mucilage; a mineral oredible oil or fatty acids; or a macrogel. Such compositions mayadditionally comprise suitable surface active agents such assurfactants, and suspending agents such as agar, vegetable gums,cellulose derivatives, and other ingredients such as preservatives,antioxidants, and the like.

The compositions can also be formulated for injection into the subject.For injection, the compositions of the invention can be formulated inaqueous solutions such as water or alcohol, or in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. The solution may contain formulatory agentssuch as suspending, stabilizing and/or dispersing agents. Injectionformulations may also be prepared as solid form preparations which areintended to be converted, shortly before use, to liquid formpreparations suitable for injection, for example, by constitution with asuitable vehicle, such as sterile water, saline solution, or alcohol,before use.

The compositions can also be formulated in sustained release vehicles ordepot preparations. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may beformulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt. Liposomes and emulsions are well-known examples of deliveryvehicles suitable for use as carriers for hydrophobic drugs.

Administration of the compositions can be by infusion or injection(intravenously, intramuscularly, intracutaneously, subcutaneously,intrathecal, intraduodenally, intraperitoneally, and the like). Thecompositions can also be administered intranasally, vaginally, rectally,orally, or transdermally. Preferably, the compositions are administeredintravenously. Administration can be at the direction of a physician.

For buccal administration, the compositions may take the form oftablets, troche or lozenge formulated in conventional manner.Compositions for oral or buccal administration, may be formulated togive controlled release of the active compound. Such formulations mayinclude one or more sustained-release agents known in the art, such asglyceryl mono-stearate, glyceryl distearate and wax.

Compositions may be applied topically. Such administrations includeapplying the compositions externally to the epidermis, the mouth cavity,eye, ear and nose. This contrasts with systemic administration achievedby oral, intravenous, intraperitoneal and intramuscular delivery.Compositions for use in topical administration include, e.g., liquid orgel preparations suitable for penetration through the skin such ascreams, liniments, lotions, ointments or pastes, and drops suitable fordelivery to the eye, ear or nose.

Various alternative pharmaceutical delivery systems may be employed.Non-limiting examples of such systems include liposomes and emulsions.Certain organic solvents such as dimethylsulfoxide also may be employed.Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid polymers containing thetherapeutic agent. The various sustained-release materials available arewell known by those skilled in the art. Sustained-release capsules may,depending on their chemical nature, release the compounds over a rangeof several days to several weeks to several months.

Induction of cell differentiation is also stimulated by administeringpeptide fragments of angiocidin comprising the amino acid sequence ofSEQ ID NO:2 or SEQ ID NO:3, and mimetic peptides exhibiting theseeffects. These therapeutic peptides can be formulated and administeredas described above.

The following examples are provided to describe the invention in greaterdetail. The examples are intended to illustrate, not to limit, theinvention.

Example 1 Preparation of Recombinant Angiocidin

Purified, recombinant, his-tagged angiocidin was expressed in bacteriaand purified as described in Zhou, et al., J. Cell Biochem. 92: 125-146,2004.

Example 2 Angiocidin Induces Differentiation of Monocytes to Macrophages

THP-1 cells, a monocytic leukemic human cell line (ATCC No. TIB-202),were cultured and treated with angiocidin or with conditioned mediumfrom monocyte and T-cell cultures as described in Kremlev, et al., J.Neuroimmunol. 194: 132-142, 2008 (“Kremlev”). Treatment with 1.0 μg/mLangiocidin for 24 h induced a dramatic morphological change in the THP-1cells as shown in FIG. 1. Normally, THP-1 cells grow in suspension anddo not attach to a culture surface. However, angiocidin-treated cellsbecame adherent, spread on plastic and on matrix proteins, e.g.,collagen, and developed focal adhesion contacts containing vinculin(Kremlev).

The ability of angiocidin to induce these phenotypic changes requiresthe 20 amino acid sequence of SEQ ID NO:2. This 20 amino acid sequencebinds to a2131 integrin (Sabherwal, et al., Exp. Cell. Res. 312:2443-2453, 2006). A deletion mutant of angiocidin lacking this sequencefailed to induce morphological changes. However, a 25 amino acid peptidecomprising these 20 amino acids was capable of inducing the phenotypicchanges observed after treatment with the full angiocidin protein(Kremlev).

Treatment with conditioned medium from angiocidin-treated THP-1 cellcultures (from which free angiocidin was removed by immunoadsorbtion)also stimulated THP-1 cell adhesion and cell migration as shown in FIG.2, (methods described in Kremlev), suggesting that angiocidin treatmentstimulates the production of factors that induce cell differentiation.

Angiocidin-treated THP-1 cells developed focal adhesions and exhibitedan increase in phosphorylated FAK protein and phosphorylated paxillin.Angiocidin also stimulated mRNA expression for a number of proteins thatare “differentiation state dependent,” including CD36, MARCO, CD14,CD69, and a2-macroglobulin (FIG. 3). Angiocidin treatment also increasedthe concentration of specific inflammatory cytokines and chemokinessecreted into the culture medium as shown in FIG. 4. Experimentalprocedures underlying the results shown in FIGS. 3 and 4 were derivedfrom Gaurnier-Hausser, et al., Cancer Research 68: 5905-5914, 2008.

Monocyte to macrophage differentiation is characterized by an increasein phagocytic activity (Reyes, et al., Infect. Immun. 67: 3188-3192,1999). Similarly, angiocidin-treated THP-1 cells acquired increasedphagocytic activity upon differentiation. Furthermore, angiocidintreatment stimulated THP-1 cells to secrete active matrixmetalloproteinase 9 (MMP-9) in the same manner as activated macrophages.(Gaurnier-Hausser, et al., Cancer Res. 68: 5905-5914, 2008).

These results demonstrate that angiocidin can induce differentiation ofmonocytes in human blood and suggest that angiocidin could be used toinhibit tumor cell proliferation and secondary metastasis and in thetreatment of leukemia.

Example 3

Immune stimulation of T-lymphocytes from human blood Human peripheralblood mononuclear cells (PBMC) were collected, and mononuclear (MNP) andT-cell enriched cell populations were obtained and cultured as describedin Kremlev. Cell cultures were treated with angiocidin at 1.0 μg/mLovernight. Conditioned medium from treated cultures was collected andanalyzed on a membrane array of cytokines obtained from RayBiotech, Inc.(RAYBIO® Human Cytokine Antibody Array 3) as described in Kremlev.Angiocidin treatment activated CD4+ and CD8+ T-lymphocytes andstimulated the synthesis and secretion of inflammatory cytokines inT-cell and MNP cell populations (Kremlev). These results show thatangiocidin is a potent systemic immune stimulator that can induceinflammatory signals in autoimmune diseases, such as multiple sclerosis(MS).

Example 4 Angiocidin Facilitates Antigen Presentation by MNP Cells toT-Lymphocytes

Transcription of RNA for proteins important in antigen presentation wasincreased in THP-1 cells after angiocidin treatment (Kremlev). Theseproteins included HLA-DPB2, HLA-DPB1, and B7-2. Therefore, the effectsof angiocidin on antigen presentation were examined.

Peripheral blood mononuclear cells (PBMC) were plated on tissue culturedishes. The adherent monocyte fraction was treated with 1.0 μg/mLangiocidin for seven days in the presence or absence of the antigen,human myelin basic protein peptide (MBP) at 40 ug/mL. The non-adherentPMBC fraction, which contained T-cells, was separately treated withangiocidin in the presence and absence of MBP. The culture medium wasthen removed from the cultured monocytes and the cells were challengedwith nonadherent PBMC. Challenging PBMC comprised three groups:untreated (control) cells; MBP-treated cells, and MBP+angiocidin-treatedcells. Conditioned medium was collected at 24 and 48 h and analyzed forIL-2 by ELISA. Methods and results are described in detail in Kremlev.

As shown in FIG. 5, a significant increase in IL-2 production was foundin angiocidin+MBP treated monocytes challenged by angiocidin+MBP-treatedPBMC after 24 and 48 h in culture (FIG. 5D). A significant, but smaller,increase in IL-2 production was also observed in angiocidin+MBP-treatedmonocytes challenged with MPB-treated PBMC (FIG. 5D).

These results demonstrate that angiocidin promotes antigen presentationand increases the ability of antigen-specific T-effector lymphocytes toproduce IL-2, which is a marker for proliferating T-cells presented withantigen (Robb, et al., J. Exp. Med. 154: 1455-74, 1981. IL-2 is referredto in this paper as TCGF).

Example 5 Angiocidin is Expressed in MS Lesions of MS Patients

Immunohistochemistry was performed on sections of brain from 14 patientswith MS as described in Kremlev. Prominent angiocidin expression andIL-7 expression was observed in astrocytes and the surroundingextracellular matrix within demyelinated plaques in all 14 patients asshown in FIG. 1 of Kremlev. Normal tissue in adjacent areas showed noexpression of angiocidin. These results, taken with the results fromExamples 3 and 4 suggest that angiocidin and its downstream mediatorsare important target molecules in the treatment of MS.

Example 6 Induction of Differentiation of Blastomere-Like Stem Cellsfrom Human Blood

Blastomere-like stem cells (BLSC) (www.moragabiotech.com/techblscs.htm)are small (less than 2 μm diameter) totipotent stem cells that wereisolated from human blood and provided by Moraga BiotechnologyCorporation. BLSC proliferate as undifferentiated spheroidal cells insuspension when plated on collagen-coated tissue culture dishes. Howeverwithin 24 h of the addition of 10 μg/mL angiocidin to the medium, mostof the cells adhere to collagen in coated tissue culture dishes and showenhanced growth potential as evidenced by the accumulation of largeaggregates (FIG. 6A, arrows). Angiocidin has the ability to induceproliferation in both adherent and non-adherent subpopulations of BLSC.Angiocidin has an equal or greater effect on the adherent subpopulationof BLSC as bFGF and EGF, potent stem cell growth factors, in aggregatingBLSC on collagen-coated dishes when added at 20 ng/mL (FIG. 6B).However, the combination of bFGF and angiocidin at 20 ng/mL and 10μg/mL, respectively, elicits similar effects on BLSC as angiocidin alone(FIG. 6C, arrow).

These results demonstrate that angiocidin has potential in stimulatingproliferation of blood-derived stem cells, suggesting that angiocidincould be used as a general inductant in combination with specific stemcell growth factors to produce differentiated cells for cell and tissueregeneration during tissue repair. These results suggest important usesfor angiocidin not only in the health care industry, but also in thecosmetic industry.

Example 7 Inhibition of Melanoma Stem Cell Proliferation

Stem cells capable of developing into melanoma cells were obtained fromDr. Meenhard Herlyn and are described in Fang, et al., Cancer Research65: 9328-9337, 2005. The cells were plated in 8-well chamber slideseither uncoated, or coated with 2 μg fibronectin or Type 1 collagen(Fisher Scientific), and grown in mesenchymal stem cell mediasupplemented with 5 ng/mL fibroblast growth factor (Invitrogenmesenchymal stem cell media or Moraga stem cell media). Type I collagen(1 mg/mL) was dissolved in 0.1N acetic acid and each well was coatedwith 2 μg of the collagen solution by evaporating 100 μL of a 30%ethanol solution containing 2 μL of the 1 μg/mL stock solution per wellof the chamber. After one and two weeks, cell number was assessedcolorimetrically using the Almar Blue assay (Biosource). As shown inFIG. 7, 50 μg/mL of angiocidin treatment inhibited cell proliferation byabout 60-90%. These results demonstrate that angiocidin is a potentinhibitor of melanoma stem cell proliferation and suggest thatangiocidin would be an effective therapy for inhibiting melanoma.

Example 8 Angiocidin Promotes Neurite Outgrowth

PC12 is a cell line derived from a pheochromocytoma of the rat adrenalmedulla (Greene L A, Tischler A S (July 1976). “Establishment of anoradrenergic clonal line of rat adrenal pheochromocytoma cells whichrespond to nerve growth factor”. Proc. Natl. Acad. Sci. U.S.A. 73 (7):2424-8). PC12 cells stop dividing and terminally differentiate whentreated with nerve growth factor (NGF). This makes PC12 cells useful asa model system for neuronal differentiation. It was observed thatangiocidin promotes NGF-mediated differentiation of PC12 cells (FIG. 8).

FIG. 8 shows the effect of angiocidin on PC12 neurite outgrowth.Briefly, PC-12 cells were plated in six well plates at a density of2.5×105 cells/mL in 1 mL of Dulbecco's Modified Eagle Medium (DMEM)containing 1% horse serum and 1% Penicillin and streptomycin, treatedwith 100 ng/mL of NGF, 1 ug/nnL of recombinant angiocidin, or both andallowed to incubate at 37° for 72 hours. After 72 hours, pictures weretaken of five random fields at 400× using Hoffman lens. Neuriteoutgrowth was quantitated using Image 3 software, a public source openaccess program. To be considered a neurite, a process had to meet thefollowing criteria: (1) Neurite had to be at least 10 um in length; (2)Neurite had to start and end within the field of vision; and (3)Neurites with branches were measured to the branching point and theneach branch was measured separately.

Example 9 Angiocidin Inhibits Glioma Stem Cell Growth

FIG. 9 shows the effect of angiocidin on growth of glioma stem cells.This figure shows that when glioma stem cells are cultured for eightdays in stem cell media consisting of Dulbecco's Modified Eagle'sMedium/Nutrient F-12 Ham containing 100 ng/ml basic fibroblast growthfactor, they self-renew forming a multicellular spheroidal cell masscontaining approximately 50-100 stem cells. However, when these cellswere cultured in stem cell media containing 2 μg/ml angiocidin,proliferation was completely inhibited and eventually the cells died.

Cells were viewed under phase contrast microscopy at 200× magnification.Briefly, glioma stem cells were isolated from a resected tumor obtainedfrom a patient with glioma multiforma (GBM). Tumor tissue wasdissociated in phosphate buffered saline by treatment of minced tumortissue with collagenase (1 mg/ml) for one hour. Cells were harvested bycentrifugation and the cells plated in stem cell media in a six wellplate coated with mouse anti-CD33 IgG (coated by incubating the plateswith 1 μg/ml anti-CD33 IgG overnight followed by one rinse in phosphatebuffered saline containing 1 mg/ml bovine serum albumin). After one hourincubation, adherent cells were removed by scraping and 50 cells wereplated into six well culture dishes in stem cell media with and without2 μg/ml angiocidin. Growth of cells was monitored for eight days. FIG. 9shows a representative high power field from untreated andangiocidin-treated cells.

Example 10 Angiocidin Binds Basic Fibroblast Growth Factor (FGF-2) andNerve Growth Factor (NGF)

Since neurite outgrowth and stem cell renewal is dependent on growthfactors such as NGF and FGF-2, whether angiocidin binds to these growthfactors was investigated. A simple binding assay was developed, in whichthe growth factors are adsorbed to the wells of a micotiter dish and theadsorbed factors are allowed to bind with biotinylated angiocidin. Thebound biotinylated angiocidin was detected with streptavidin-coupledhorse radish peroxidase and developed with the colorimetric substrateultra 3,3′,5,5′-tetramethylbenzidine (TMB) (FIG. 10A). To determine thatbiotinylated angiocidin binds specifically, we were able to show thatbinding was competed with unbiotinylated angiocidin (FIG. 10B).

As shown in FIG. 10, angiocidin directly binds to adsorbed NGF andFGF-2. Briefly, in a 96-well plate, 1 ng of NGF or FGF-2 in a 20 mMHEPES buffer was adsorbed to the well in duplicate overnight at 4° C.Remaining buffer was aspirated and the wells were washed withphosphate-buffered saline (PBS). Then, the plate was blocked in 1% (w/v)BSA in PBS for 30 minutes without shaking. Aliquots in a 20 mM HEPESbuffer containing various dilutions of biotinylated angiocidin preparedaccording to Sabherwal Y., Rothman, V. L., Svetoslav, D, L'Heureax D.Z., Marcinkiewicz, C., Sharma, M., and Tuszynski, G. P., Integrin α2β1mediates the anti-angiogenic and anti-tumor activities of angiocidin, anovel tumor-associated protein, Experimental Cell Research, 312:2443-2453, 2006 were added to the wells. The 96 well plate was incubatedat room temperature with shaking for two hours. Remaining proteinsolutions were aspirated and then the plate was washed withtris-buffered saline containing 0.1% Tween 20 (TBST) three times. Analiquot of 100 μl of a 0.05 μg/ml streptavidin horseradish peroxidasesolution dilution in TBST was added for 30 minutes with shaking.Remaining reagent was then aspirated and the wells were washed threetimes with TBST. Ultra 3,3′,5,5′-tetramethylbenzidine (TMB) was added ineach well for 15 minutes and 100 μl of 0.5M H2504 was added to stop thereaction and the plate read in an ELISA plate reader at 450 nm. Forcompetition experiments shown in FIG. 10B, non-biotinylated protein wasadded to 100 ng/ml biotinylated angiocidin and binding was performed ineither FGF-2 or NGF-coated plates as described above.

Example 11 Angiocidin and NGF Induce Cell Arrest and Apoptosis in MDAMB231 Breast Carcinoma Cells

To determine if angiocidin growth factor complex plays any role tumorcell proliferation, breast cancer cells, which are known to possessreceptors for NGF were treated for 48 hours with either serum-free mediacontaining 1 mg/ml BSA, or media-free media containing 1 mg/ml BSA witheither 10 μg/ml of angiocidin, 10 μg/ml angiocidin plus 100 ng/ml NGF,or 100 ng/ml NGF. Total viable cells were then determined using theAlmar blue assay as previously described in Sabherwal Y., Rothman, V.L., Svetoslav, D, L′Heureax D. Z., Marcinkiewicz, C., Sharma, M., andTuszynski, G. P., Integrin α2β1 mediates the anti-angiogenic andanti-tumor activities of angiocidin, a novel tumor-associated protein,Experimental Cell Research, 312: 2443-2453, 2006. The data in FIG. 11show that NGF and angiocidin induce growth arrest and cell death after48 hours.

These data show that angiocidin in the presence of NGF promotesdifferentiation of PC12 cells and induces cell death of breast cancercells. Additionally, angiocidin causes growth arrest of glioma stemcells presumably by its ability to complex FGF-2. Since angiocidin bindsNGF and FGF-2, we propose that angiocidin-growth factor complex inhibitsstem cell growth by withdrawing growth factor from the cells whileangiocidin NGF complexes promote cell differentiation while promotingtumor cell growth arrest and apoptosis.

Example 12 Effect of Angiocidin on Growth of Human Breast Tumors

In FIG. 12, the data show that MDA-MB-231 human breast tumor cellstransfected with angiocidin to over-express angiocidin formed smallercolonies in soft agar and developed smaller tumors when injectedsubcutaneously into athymic mice.

Example 13 Angiocidin-Transfected MDA-MB-231 Tumor Cells Develop SmallerTumors in Mice and Smaller Colonies in Soft Agar

MDA-MB-231 breast cancer cells were transfected with angiocidin usingthe pcDNA3.1 transfection system as described by the manufacturer. Atleast two angiocidin over-expressing clones were selected: sense 1 and2, as well as the vector control. In FIG. 12, the panels marked senseshow the sense 1 clone. Similar results were obtained with sense 2clones. Panel A shows paraformaldehyde-fixed cells grown in culture andstained with anti-angiocidin antibody and developed with DAB (100×).Panel B shows Western blot of cells grown in panel A. Panel C showscolonies grown in soft agar as seen by phase contrast phase contrast(50× mag). Panel D shows tumor volumes of cells grown in mice (n=6 pergroup±std). Athymic mice were injected with 1 million cellssubcutaneously and tumors allowed to develop for three weeks. Tumorvolumes were measured as previously described (3. Zhou, V. L. Rothman,I. Sargiannidou, S. Dimitrov, C. Qiu, E. Smith, 3. Sheffield, M. Sharma,G. P. Tuszynski, Cloning and characterization of angiocidin, a tumorcell binding protein for thrombospondin-1, J Cell Biochem 92 (2004)125-146).

Example 14 The Effect of Angiocidin on Differentiation of Leukemia StemCells

The CD34+/CD38− stem cell subset of the AML cell line THP-1 was recentlyisolated by magnetic bead cell sorting. These cells were cultured inserum-free media in the absence and presence of 10 μg/ml of angiocidinfor 48 hours. Cells in the absence of angiocidin remained rounded whilethose in the presence of angiocidin all became adherent and spread (FIG.13). The angiocidin-differentiated cells were morphologicallyindistinguishable from the parental mixed population that wasdifferentiated by angiocidin. These results strongly suggest thatangiocidin may be targeting the leukemic stem cell population andfurther underscore the high clinical impact of angiocidin as a potentialtherapeutic for the treatment of leukemia.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

1. A method of treating a glioma in a subject, comprising administeringto the subject an amount of angiocidin effective to treat the glioma. 2.The method of claim 1, wherein angiocidin is administered in acomposition comprising angiocidin and a pharmaceutically acceptablecarrier.
 3. The method of claim 2, wherein angiocidin is administered tothe subject in a dose of about 0.001 to 10 mg/kg body weight.
 4. Themethod of claim 1, further comprising administering to the subject aneffective amount of fibroblast growth factor-2, nerve growth factor,brain-derived neural factor, neurotropin-3, epidermal growth factor, orstem cell growth factor.
 5. The method of claim 4, wherein theangiocidin binds to the fibroblast growth factor-2, nerve growth factor,brain-derived neural factor, neurotropin-3, epidermal growth factor, orstem cell growth factor to form a complex, and wherein the complexinduces growth arrest of the glioma cells.
 6. The method of claim 4,wherein the angiocidin binds to the fibroblast growth factor-2, nervegrowth factor, brain-derived neural factor, neurotropin-3, epidermalgrowth factor, or stem cell growth factor to form a complex, and whereinthe complex promotes healthy stem cell differentiation at or proximal tothe site of the glioma.
 7. A method of treating breast cancer in asubject, comprising administering to the subject an amount of angiocidineffective to treat the breast cancer.
 8. The method of claim 7, whereinangiocidin is administered in a composition comprising angiocidin and apharmaceutically acceptable carrier.
 9. The method of claim 8, whereinangiocidin is administered to the subject in a dose of about 0.001 to 10mg/kg body weight.
 10. The method of claim 7, further comprisingadministering to the subject an effective amount of fibroblast growthfactor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor. 11.The method of claim 10, wherein the angiocidin binds to the fibroblastgrowth factor-2, nerve growth factor, brain-derived neural factor,neurotropin-3, epidermal growth factor, or stem cell growth factor toform a complex, and wherein the complex induces growth arrest of thebreast cancer cells.
 12. The method of claim 10, wherein the angiocidinbinds to the fibroblast growth factor-2, nerve growth factor,brain-derived neural factor, neurotropin-3, epidermal growth factor, orstem cell growth factor to form a complex, and wherein the complexpromotes healthy stem cell differentiation at or proximal to the site ofthe breast cancer.
 13. (canceled)
 14. (canceled)
 15. (canceled) 16.(canceled)
 17. (canceled)
 18. A method of arresting growth of a tumorcell, comprising contacting the tumor cell with an amount of angiocidineffective to arrest growth of the tumor cell.
 19. The method of claim18, wherein the tumor cell undergoes apopotosis.
 20. The method of claim18, wherein the tumor cell is a glioma cell.
 21. The method of claim 18,wherein the tumor cell is a breast cancer cell.
 22. The method of claim18, wherein the tumor cell is a leukemia cell.
 23. The method of claim18, wherein the tumor cell is a melanoma cell.
 24. The method of claim18, wherein the angiocidin is complexed with fibroblast growth factor-2,nerve growth factor, brain-derived neural factor, neurotropin-3,epidermal growth factor, or stem cell growth factor.
 25. A method ofinducing differentiation of a leukemia cell, comprising contacting theleukemia cell with an amount of angiocidin effective to inducedifferentiation of the leukemia cell.
 26. The method of claim 25,wherein the differentiation inhibits the tumorigenicity of the leukemiacell.
 27. The method of claim 25, wherein the differentiation inducesthe leukemia cell to confer a non-cancerous phenotype.
 28. A method fortreating a tumor having a receptor for fibroblast growth factor-2, nervegrowth factor, brain-derived neural factor, neurotropin-3, epidermalgrowth factor, or stem cell growth factor in a subject, comprisingadministering to the subject an amount of angiocidin effective to treatthe tumor expressing a receptor for fibroblast growth factor-2 or nervegrowth factor.
 29. The method of claim 28, wherein angiocidin isadministered in a composition comprising angiocidin and apharmaceutically acceptable carrier.
 30. The method of claim 28, whereinangiocidin is administered to the subject in a dose of about 0.001 to 10mg/kg body weight.
 31. The method of claim 28, wherein the tumorexpressing a receptor for fibroblast growth factor-2, nerve growthfactor, brain-derived neural factor, neurotropin-3, epidermal growthfactor, or stem cell growth factor is glioma, breast cancer, leukemia,or melanoma.